The present invention relates to aminoalkylphenols. More particularly, the present invention relates to aminoalkylphenol derivatives of formula 1
wherein:
R1 is hydrogen, loweralkyl, a group of the formula CONR6R7, a group of the formula CH2COOR8, a group of the formula CH2CH2OH, a group of the formula CH2CN, or a group of the formula CH2C≈Cxe2x80x94R9, a group of the formula 
xe2x80x83or a group of the formula Si(R11)3;
R2 is hydrogen, loweralkyl, a group of the formula CONR6R7, a group of the formula SO2R10, a group of the formula 
xe2x80x83or a group of the formula Si(R11)3;
R3 is hydrogen or loweralkyl;
R4 is hydrogen or loweralkyl;
R5 is hydrogen, loweralkyl, a group of the formula 
xe2x80x83or a group of the formula 
R4 and R5 taken together with the nitrogen atom to which they are attached form a group of the formula 
xe2x80x83group of the formula 
xe2x80x83a group of the formula 
xe2x80x83a group of the formula 
R6 is hydrogen or loweralkyl;
R7 is alkyl of 1 to 10 carbon atoms, or a group of the formula 
xe2x80x83a group of the formula 
xe2x80x83or a group of the formula 
R6 and R7 taken together with the nitrogen atom to which they are attached form a group of the formula 
R8 is loweralkyl;
R9 is hydrogen, a group of the formula C(R14R14)OH, a group of the formula 
xe2x80x83group of the formula 
R10 is loweralkyl;
R11 is loweralkyl;
R12 is loweralkyl, hydroxyloweralkyl, a group of the formula COR15, a group of the formula 
xe2x80x83a group of the formula 
xe2x80x83a group of the formula 
xe2x80x83a group of the formula 
xe2x80x83or a group of the formula 
R13 is hydrogen or loweralkyl;
R14 is hydrogen or loweralkyl;
R14 is hydrogen or loweralkyl;
R15 is a group of the formula 
xe2x80x83or loweralkyl;
R20 is loweralkyl;
X is hydrogen or halogen;
Z is hydrogen, halogen, loweralkyl, hydroxyl, loweralkoxy, trifluoromethyl, nitro or cyano, R20CO, or a group of the formula OCONR6R7;
m is 1 or 2;
n is 0 or 1;
p is 1 or 2;
q is 0, 1 or 2;
r is 0, 1 or 2;
the optical isomers thereof; or the pharmaceutically acceptable salts thereof, which are useful for relieving memory dysfunction and thus indicated in the treatment of Alzheimer""s disease, alone, or in combination with adjuvants.
Subgeneric thereto are compounds wherein:
(a) R1 is a group of the formula CONR6R7;
(b) R2 is loweralkyl;
(c) R2 is a group of the formula CONR6R7;
(d) R1 is loweralkyl;
(e) R4 and R5 taken together with the nitrogen to which they are attached form a group of the formula 
(f) R1 is a group of the formula CONR6R7 and R4 and R5 together with the nitrogen to which they are attached form a group of the formula 
xe2x80x83and n is 0; and
(g) R2 is a group of the formula CONR6R7 and R4 and R5 taken together with the nitrogen to which they are attached form a group of the formula 
xe2x80x83and n is 0.
The present invention also relates to compounds of formula 2
wherein R16 and R17 are independently hydrogen or alkyl of 1 to 10 carbon atoms and R18 is a group of the formula 
wherein W is hydrogen, loweralkyl, loweralkoxy, hydroxy, halogen or trifluoromethyl, and s is 1 or 2; X is hydrogen or halogen; m is 1 or 2, the optical isomers thereof; or the pharmaceutically acceptable salts thereof; of formula 3
wherein R1 is loweralkyl, benzyl, or a group of the formula CH2C≈CH; or a group of the formula CONR6R7 wherein R6 is hydrogen and R7 is loweralkyl; R2 is hydrogen, loweralkyl, a group of the formula Si(R11), wherein R11 is loweralkyl or R2 is a group of the formula COR6R7 wherein R6 is hydrogen and R7 is loweralkyl; X is hydrogen or halogen; m is 1 or 2; the optical isomers thereof; or salts thereof; of formula 4
wherein R1 is loweralkyl, a group of the formula C≈CH, a group of the formula CH2COOR8, a group of the formula 
a group of the formula CONR6R7 or a group of the formula Si(R11)3; R2 is a group of the formula SO2R10, a group of the formula 
a group of the formula Si(R11)3 or a group of the formula CONR6R7; R8 is loweralkyl; Z is hydrogen, halogen, loweralkyl, hydroxyl, loweralkoxy, trifluoromethyl, nitro or cyano; p is 1 or 2; R6 is hydrogen; R7 is a group of the formula 
wherein R13 is loweralkyl and Z and p are as defined herein; and
R11 is loweralkyl; X is hydrogen or halogen; m is 1 or 2, or the optical isomers thereof; and formula 5
wherein R2 is hydrogen, loweralkyl or a group of the formula CONR7R7 wherein R6 and R7 are loweralkyl; R3 is hydrogen or loweralkyl; R4 and R5 taken together with the nitrogen atom to which they are attached form a group of the formula 
wherein R12 is a group of the formula 
n is 0; X is hydrogen or halogen; m is 1 or 2 or the optical isomers thereof; or pharmaceutically acceptable salts thereof; which are useful as intermediates for the preparation of the aminoalkyl phenols of the present invention and for relieving memory dysfunction.
The invention also includes compounds of formula 43
wherein:
R2 is hydrogen, loweralkyl or a group of the formula CONR6R7;
R3 is hydrogen or loweralkyl;
R4 and R5 taken together with the nitrogen atom to which they are attached form a group of the formula 
R12 is a group of the formula 
Z is hydrogen, halogen, loweralkyl, hydroxyl, loweralkoxy, trifluoromethyl, nitro, cyano or COCH3;
m is 1 or 2;
n is 0 or 1;
p is 1 or 2;
the optical isomers thereof, or the pharmaceutically acceptable salts thereof.
As used throughout the specification and appended claims, the term xe2x80x9calkylxe2x80x9d refers to a straight or branched chain hydrocarbon radical containing no unsaturation and having 1 to 12 carbon atoms. Examples of alkyl groups are methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 1-pentyl, 3-hexyl, 4-heptyl, 2-octyl, 3-nonyl, 4-decyl, undecyl, dodecyl, and the like. The term xe2x80x9calkanolxe2x80x9d refers to a compound formed by a combination of an alkyl group and hydroxy radical. Examples of alkanols are methanol, ethanol, 1- and 2-propanol, 2,2-dimethylethanol, hexanol, octanol, decanol and the like. The term xe2x80x9calkanoic acidxe2x80x9d refers to a compound formed by combination of a carboxyl group with a hydrogen atom or alkyl group. Examples of alkanoic acids are formic acid, acetic acid, propanoic acid, 2,2-dimethylacetic acid, hexanoic acid, octanoic acid, decanoic acid and the like. The term xe2x80x9chalogenxe2x80x9d refers to a member of the family fluorine, chlorine, bromine, or iodine. The term xe2x80x9calkanoylxe2x80x9d refers to the radical formed by removal of the hydroxyl function from an alkanoic acid. Examples of alkanoyl groups are formyl, acetyl, propionyl, 2,2-dimethylacetyl, hexanoyl, octanoyl, decanoyl and the like. The term xe2x80x9calkoxyxe2x80x9d refers to a monovalent substituent which consists of an alkyl group linked through an ether oxygen and having its free valence from the ether oxygen. Examples of alkoxy groups are methoxy, ethoxy, proproxy, 2,2-dimethylethoxy, hexoxy, octoxy, decoxy and the like. The term xe2x80x9clowerxe2x80x9d as applied to any of the aforementioned groups refers to a group having a carbon skeleton containing up to and including 10 carbon atoms.
The compounds of the present invention which lack an element of symmetry exist as optical antipodes and as the racemic forms thereof. The optical antipodes may be prepared from the corresponding racemic forms by standard optical resolution techniques, involving, for example, the separation of diastereomeric salts of those instant compounds characterized by the presence of a basic amino group and an optically active acid, those instant compounds characterized by the presence of a carboxyl acid group and an optically active base, or by synthesis from optically active precursors.
The present invention comprehends all optical isomers and racemic forms thereof of the compounds disclosed and claimed herein. The formulas of the compounds shown herein are intended to encompass all possible optical isomers of the compounds so depicted.
The novel aminoalkylphenols of the present invention are prepared by the processes illustrated in the Reaction Schemes. To prepare an aminoalkylphenol 1 wherein R3 is hydrogen or loweralkyl and n is 0, a benzaldehyde 6, or a phenylalkylketone 10, is condensed with an amine 7 in the presence of a reducing agent to provide 8 or 11, respectively. The reductive amination is generally performed in the presence of a mild selective reducing agent such as an alkali metal trialkanoyloxyborohydrideor an alkali metal cyanoborohydride in a suitable solvent. Among alkali metal trialkanoyloxy-borohydrides, there may be mentioned lithium-, sodium- and potassium triacetoxyborohydride. Among alkali metal cyanoborohydrides, there may be mentioned lithium-, sodium- and potassium cyanoborohydride. Among suitable solvents, there may be mentioned halocarbons such as dichloromethane, trichloromethane, 1,2-dichloroethane and 1,1-dichloroethane, or ethereal solvents such as tetrahydrofuran, dioxane and 2-methoxyethyl ether, optionally containing an alkanoic acid such as acetic acid when a trialkanoyloxyborohydride is employed, and an alkanol such as methanol or ethanol in the presence of a mineral acid such as hydrochloride acid or an alkanoic acid such as acetic acid when a cyanoborohydride is employed. Sodium triacetoxyborohydride is the preferred reducing agent; 1,2-dichloroethane is the preferred halocarbon. The reaction of a benzaldehyde 6 and a secondary amine 7 is preferably carried out in the absence of an alkanoic acid such as acetic acid. While the temperature at which the reaction is conducted is not narrowly critical, the reaction is conveniently carried out at ambient temperature.
Alternatively, an aminoalkylphenol 1 wherein R3 is hydrogen and n is 0, is prepared by condensing a benzyl halide 9 with an amine 7 to provide 13. The condensation is accomplished by means of an alkali metal hydride such as sodium hydride in a halocarbon such as chloromethane, dichloromethane, or 1,1- or 1,2-dichloroethane or dimethylformamide, at about ambient temperature, although reduced or elevated temperatures may be employed. The preferred condensation medium is sodium hydride as a dispersion in oil in dichloromethane.
To fabricate an aminoalkylphenol derivative 1 wherein R3 is hydrogen or loweralkyl and n is 1, a haloethylphenol 12 is condensed with an amine 7 to provide 13 by the processes herein described.
To gain access to an aminoalkylphenol derivative 1 wherein R3 is loweralkyl and n is 0, a phenylalkylketone 10 is condensed with an amine 7 to provide 11. The reaction is carried out in the presence of a reducing agent such as titanium (IV) alkoxide in a suitable solvent such as acetonitrile or dichloromethane at about ambient temperature followed by an alkali metal cyanoborohydride in an alkanol, or an alkanoic acid such as acetic acid or a mineral acid such as hydrochloric acid, optimally an alkanoic acid also at ambient temperature. Among titanium (IV) alkoxides there may be mentioned titanium (IV) methoxide, titanium (IV) ethoxide, titanium (IV) 2-propoxide, and titanium (IV) 1-propoxide. Among alkali metal cyanoborohydrides there may be mentioned lithium cyanoborohydride, sodium cyanoborohydride and potassium cyanoborohydride. Among alkanols, there may be mentioned methanol, ethanol, 1-propanol and 2-propanol. The preferred reagents for effecting the reductive condensation are titanium (IV) 2-propoxide, sodium cyanoborohydride, ethanol and dichloromethane.
The aminoalkylphenol derivatives and related compounds thereof of the present invention of formula 1 having the described functionality on the benzene ring and in the side-chain, i.e., compounds of formula 1 wherein R1, R2, R3, R4, R5, X, m and n are as hereinbefore disclosed may be prepared starting from benzaldehydes 6 phenylalkyl ketones 10, benzylhalides 9, or phenylethylhalides 12 having the described functionality intact or from the aminoalkylphenol derivatives 8, 11, or 13 by manipulation of the functionality thereof.
To convert an aromatic hydroxyl group (xe2x80x94OH) to a carbamoyloxy moiety 
an aminoalkylphenol 1 wherein R1 or R2 is hydrogen is treated-with, for example, a carbamoyl halide 14 of the formula
R6R7NCOHalxe2x80x83xe2x80x8314
wherein R6 and R7 are as herein defined and Hal is bromo or chloro in the presence of an acid acceptor such as an alkali metal carbonate, e.g., lithium-, sodium-, potassium- or cesium carbonate in a suitable solvent such as acetonitrile or dichloromethane, or combinations thereof, at about ambient temperature. Cesium carbonate is the preferred acid acceptor.
Alternatively, a tertiary amine such as 1,8-diazabicyclo[5.4.0]undec-7-ene may be used as the acid acceptor and acetonitrile as the solvent in the reaction of a phenol 1 with a carbamoyl halide 14.
The conversion of a hydroxyl group (xe2x80x94OH) to a carbamoyloxy moiety 
may also be effected by treating an aminoalkylphenol 1 wherein R1 or R2 is hydrogen with an isocyanate 15
R6 or R7xe2x80x94Nxe2x95x90Cxe2x95x90Oxe2x80x83xe2x80x8315
wherein R6 or R7 is as herein defined in the presence of a copper (I) halide, wherein the halide is chloro or bromo, in ethyl acetate, acetonitrile or dichloromethane, or combinations thereof. The introduction of a carbamoyl group 
by means of an isocyanate 15 may also be accomplished in an ethereal solvent such as tetrahydrofuran in the presence of an alkali metal carbonate such as potassium carbonate. In addition, the modification of a hydroxyl group (xe2x80x94OH), i.e., the conversion to a carbamoyloxy function 
may be effected using an amine 16
HNR6R7xe2x80x83xe2x80x8316
wherein R6 and R7 are as defined herein in the presence of 1,1∂-carbonyldiimidazole in tetrahydrofuran.
To prepare aminoalkylphenols of formula 1 wherein R1 is a group of the formula CH2C≈CR9 wherein R9 is as described herein, i.e., to introduce the ethynylalkyl moiety (CH2Cxe2x89xa0Cxe2x80x94) into the phenoxy system, an ethynylmethoxybenzaldehyde 20, which is fabricated from a benzaldehyde 17 wherein R1 is loweralkyl and X and m are as herein described by sulfonylation of 17 to an alkylsulfonylbenzaldehydel 8wherein R1 and R15 are loweralkyl, dealkylation of 18 to a hydroxybenzaldehyde 19 followed by ethynylalkylation of 19 to ethynylalkoxybenzaldehyde 20, is converted to an aminoalkylbenzene 21 wherein R3, R4, R5 and n are as described herein, which is in turn hydrolyzed to an aminoalkylphenol 22 and carbamoylated to carbamate 23 wherein R6 and R7 are as described herein. The conversion of an ethynylalkoxybenzaldehyde 20 to an aminoalkylbenzene 21 is accomplished by the methods disclosed herein. The hydrolysis of a sulfonyloxybenzene 21 to a phenol 22 is performed in an aqueous alkanol, such as aqueous methanol, containing an alkali metal hydroxide such as sodium hydroxide within a temperature range of about 25xc2x0 to 75xc2x0 C., a hydrolysis temperature of about 50xc2x0 C. being preferred. The carbamoylation of 22 to 23 is effected as herein described. Alternatively, a phenol 22 is converted to carbamate 23 by treatment with an alkali metal carbonate such as potassium carbonate in an ethereal solvent such as tetrahydrofuran followed by an isocyanate of formula 15 at ambient temperature as herein described.
Substituted ethynylmethoxybenzenecarbamates 26 wherein R9 is a group as described herein other than hydrogen are prepared from ethynylmethoxyphenols 22 by protecting the phenolic group thereof, introducing the R9 moiety to yield substituted ethynylmethoxybenzenes 25, removing the protecting group of 25 and elaborating the carbamoyloxybenzene 26 wherein R6 and R7 are as herein described.
The protection of the phenolic group of ethynylmethoxyhydroxybenzene 22 is accomplished by treating the phenol 22 with tri-(2-propyl)silyl chloride of formula 27a
((CH3)2CH)3SiClxe2x80x83xe2x80x8327a
in a dipolar aprotic solvent, e.g., dimethylacetainide, dimethylformamide, hexamethylphosphoramide or dimethylsulfoxide, dimethylformamide being preferred, in the presence of an acid acceptor such as a tertiary amine, e.g., di-(2-propyl)ethylamine at ambient temperature.
The substituent (R9), i.e., a group of the formula (R14R14xe2x80x2) CHOH wherein R14 is as described herein, or a group of the formula 
or a group of the formula 
is introduced by treating the ethynylmethoxysilyloxybenzene 24 with an alkyllithium such as n-butyllithium in an ethereal solvent such as tetrahydrofuran at a reduced temperature within the range of about xe2x88x9230xc2x0 to about xe2x88x9250xc2x0 C. followed by a carbonyl compound of formula 28a
(R14R14xe2x80x2)Cxe2x95x90Oxe2x80x83xe2x80x8328a
wherein R14 and R14xe2x80x2 are hydrogen or loweralkyl or a compound of the formula 
or a compound of the formula 
A carbamoyloxy derivative 26 of a substituted ethynylmethoxybenzene 25 is prepared in situ by treatment of 25 with tetra-n-butylammonium fluoride in an ethereal solvent such as tetrahydrofuran at ambient temperature to remove the protecting group followed by an isocyanate of formula 15 in the presence of a lithium halide, preferably lithium chloride.
Similarly, to prepare aminoalkylphenols of formula 1, i.e., compounds of formula 30 wherein Ris as herein described, a hydroxybenzaldehyde 27 is ethynylalkylated to afford ethynylalkoxyaldehyde 28 which is reductively animated to in am inoalkylethynylalkoxybenzene 30 and then alkylated to an ethynylalkoxy carbinol 30. The sequence of reactions is performed by processes herein-described.
To synthesize aminoalkylphenol derivatives of formula 1 wherein R1 is a group of the formula xe2x80x94OCH2CH2OH, i.e., hydroxyethoxyaminoalkylbenzenes,a hydroxybenzaldehyde 27 is converted to an ester 31 which is reductively aminated to an aminoalkylbenzene 32 and then reduced to hydroxyalkoxybenzene 33. The conversion of phenol 27 to ester 31 is carried out by treating 27 with an alkyl haloacetate of formula 34
HalCH2CO2R8xe2x80x83xe2x80x8334
wherein R8 is as herein defined and Hal is bromo or chloro in the presence of an acid acceptor such as an alkali metal carbonate in a suitable solvent such as acetone at an elevated temperature of about the reflux temperature of the reaction mixture. The transformation of benzaldehyde 31 to aminoalkylbenzene 32 is accomplished by methods herein described. The reduction of ester 32 is effected by treating the alkoxycarbonylalkoxybenzene32 with an alkali metal aluminum hydride such as lithium aluminum hydride in an ethereal solvent such as tetrahydrofuran at a reaction temperature of about ambient temperature.
To prepare a hydroxyethoxyaminophenol 33 wherein R2 is hydrogen for subsequent conversion to a carbamate 1 wherein R2 is carbamoyl, a hydroxyaldehyde 27 wherein R2 is benzyl is converted to a hydroxyethoxyaminoalkylbenzyloxybenzene 33 wherein R2 is benzyl by the processes described herein for the alkoxyl compounds. The benzyl group of 33 is removed by means of hydrogen at atmospheric pressure in the presence of a metal catalyst such as palladium, preferably palladium on a support, such as carbon, in an alkanoic acid such as acetic acid at ambient temperature. The introduction of the carbamoyl group may be accomplished by the processes herein described.
Removal of functional groups bound to the potential phenolic oxygen atoms of the aminoalkylbenzenes of the present invention, i.e., the formation of aminoalkylphenols of formulas 36 and 37 is effected by hydrolysis, debenzylation and/or demethylation processes. For example, to remove an aminocarbonyl group of a compound of formula 35 wherein R1 is a group of the formula CONR6R7 wherein R6 and R7 are as hereinbefore described and R2 is hydrogen, loweralkyl or benzyl, a carbamate 35 is treated with an alkali metal hydroxide in an alkanol at ambient temperature. Among alkali metal hydroxides there may be mentioned lithium, sodium or potassium hydroxide. Among alkanols there maybe mentioned methanol, ethanol or 1-propanol. Sodium hydroxide in methanol is the preferred alkali metal hydroxide; methanol is the preferred alkanol.
Similarly, removal of an alkanoyl group from an aminoalkylbenzene 35 wherein R2 is a group of the formula RCO wherein R is loweralkyl and R1 is hydrogen, loweralkyl or benzyl, is also effected by hydrolysis. In this case, the hydrolysis is achieved by treating the ester 35 with alkali metal hydroxide such as sodium hydroxide and an alkanol such as aqueous ethanol at a slightly elevated temperature of about 50xc2x0 C., although a hydrolysis temperature within the range of about ambient temperature to about the reflux temperature of the reaction medium is suitable.
To remove a benzyl group from a compound of formula 35, i.e., to debenzylate an aminoalkylbenzene 35 wherein R2 is benzyl and R1 is loweralkyl, an aminoalkylbenzene 35 is treated with ferric chloride in a halocarbon such as dichloromethane at the reflux temperature of the reaction mediums or with hydrogen in the presence of a metal catalyst such as palladium in a solvent such as acetic acid or methanol.
To remove an alkoxy group from a compound of formula 35, i.e., to dealkylate an aminoalkylbenzene 35 wherein R2 is loweralkyl, an aminoalkylbenzene 35 is treated with a hydrohalic acid such as hydrobromic acid at an elevated temperature of about 100xc2x0.
To prepare an aminoalkylpenol 40 chacterized by the presence of a heterocyclic moiety, i.e., an aminoalkyphenol 40 wherein R1, R2, R3, X, and m are as hereindescribed, a benzlamine 38 is condensed with 2-methylthio-2-imidazoline 39 in a halocarbon such as trichloromethane at about the reflux temperature of the reaction medium.
To prepare the hydrazones 42 a benzaldehyde 6 is condensed with a hydrazine 41. The condensation is carried out in a halocarbon such as dichloromethane, preferably in the
Compound of the invention of formula 43
wherein the 3-position of the benzene ring is unsubstituted are prepared by processes substanitally similar to those described of the preparation of 3-substituted phenyl compounds of the invention.
To fabricatie and aminoalkybenxene 46 wherein R2, R4 and R5 are as hereinbeforescribed and Y is halo or alkoyl, a phenyl acetic acid 44 is converted to an amide 45 which is reduced to an amine 46 methods described herein.
To prepare aminolkycarbamates 48 and 50 an aminoalkylphenol 47 is converted to cabamate 48 which is transformed to aminoalky 49 and then to carbamate 50 by methods hereindescribed. 
The aminoalkylphenol derivatives and related compounds of the present invention are useful as agents for the relief of memory dysfunction, particularly dysfunctions associated with decreased cholinergic activity such as those found in Alzheimer""s disease. Relief of memory dysfunction activity is demonstrated in the in vitro inhibition of acetylcholinesterase assay, an assay for the determination of the ability of a drug to inhibit the inactivation of acetylcholine, a neurotransmitter implicated in the etiology of memory dysfunction and Alzheimer""s dementia. In this assay, a modification of a test described G. L. Eliman, et al., Biochemical Pharmacology, 7, 88 (1961), the following reagents are prepared and employed:
1. 0.05M Phosphate Buffer (pH 7.2)
A solution of monobasic sodium phosphate monohydrate (6.85 g) in distilled water (100 ml) is added to a solution of dibasic sodium phosphate heptahydrate (13.4 g) and distilled water (100 ml) until a pH of 7.2 was attained. The solution was diluted 1 to 10 with distilled water.
2. Substrate in Buffer
The 0.05M Phosphate Buffer (pH 7.2) was added to ace tylthiocholine (198 mg) to a total volume of 100 ml, i.e., a quantity sufficient (gs) to 100 ml.
3. 5,5-Dithiobisnitrobenzoic acid in Buffer
The 0.05M Phosphate Buffer (pH 7.2) was added to 5.5-dithiobisnitrobenzoic acid to a total volume of 100 ml, i.e., a quantity sufficient (gs) to 100 ml.
4. Stock Solution of Drug
A 2 millimolar stock solution of the test drug is prepared in a quantity sufficient of either acetic acid or dimethyl sulfoxide to volume with 5,5-dithiobisnitrobenzoic acid in Buffer. Stock Solution of Drug is serially diluted (1:10) so that the final cuvette concentration is 10xe2x88x924 molar.
Male Wistar rats are decapitated, brains rapidly removed, corpora striata dissected free, weighted and homogenized in 19 volumes (approximately 7 mg protein/ml) of 0.05M Phosphate Buffer (pH 7.2) using a Potter-Elvejhem homogenizer. A 25 xcexcl aliquot of this suspension is added to 1 ml of the vehicle or various concentrations of the test drug and preincubated for 10 minutes at 37xc2x0 C. Enzyme activity is measured with a Beckman DU-50 spectrophotometer with the following software and instrument settings:
1. Kinetics Soft-Pac(trademark) Module #598273;
2. Program #6 Kindata;
3. Sourcexe2x80x94Vis;
4. Wavelengthxe2x80x94412 nm;
5. Sipperxe2x80x94none;
6. Cuvettesxe2x80x942 ml cuvettes using auto 6-sampler;
7. Blankxe2x80x941 for each substrate concentration;
8. Interval timexe2x80x9415 seconds (15 or 30 seconds for kinetics);
9. Total timexe2x80x945 minutes (5 to 10 minutes for kinetics);
10. Plotxe2x80x94yes;
11. Spanxe2x80x94autoscale;
12. Slopexe2x80x94increasing;
13. Resultsxe2x80x94yes (gives slope); and
14. Factor-1.
Reagents are added to the blank and sample cuvettes as follows:
Blank values are determined for each run to control for non-enzymatic hydrolysis of substrate and these values are automatically subtracted by the Kindata program available on kinetics soft-pac module. This program also calculates the rate of absorbance change for each cuvette.
Substrate concentration is 10 millimolar diluted 1:2 in assay yielding final concentration of 5 millimolar. 5,5-dithiobisnitrobenzoicacid concentration is 0.5 millimolar yielding 0.25 millimolar final concentration.       %    ⁢          xe2x80x83        ⁢    Inhibition    =                    Slope        ⁢                  xe2x80x83                ⁢        Control            -              Slope        ⁢                  xe2x80x83                ⁢        drug        xc3x97        100                    Slope      ⁢              xe2x80x83            ⁢      Control      
IC50 values are calculated from log-probit analysis
Relief of memory dysfunction is achieved when the present alkylaminophenol derivatives and related compounds are administered to a subject requiring such treatment as an effective oral, parenteral or intravenous dose of from 0.10 to 50 mg/kg of body weight per day. A particularly effective amount is about 10 mg/kg of body weight per day. It is to be understood, however, that for any particular subject, specific dosage regimens should be adjusted according to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compound. It is to be further understood that the dosages set forth herein are exemplary only and that they do not, to any extent, limit the scope or practice of the invention.
The alkylaminophenol derivatives and related compounds of the present invention are also useful as agents for treating depression. Depression treatment is demonstrated in the in vitro clonidine binding: xcex12-receptor assay, an assay for the determination of the ability of a drug to bind the clonidine: xcex12-receptor, performed by a modification of assays described by D. C. U""Prichard,et al., Molecular Pharmacology,16, 47(1979) and D. C. U""Prichard,et al., Molecular Pharmacology, 13, 454 (1976).
The following reagents are prepared:
1. Tris buffer, pH 7.7
a. 57.2 g Tris hydrochloride
16.2 g Tris Basexe2x80x94q.s. to 1 liter (0.5 M Tris buffer, pH 7.7)
b. Make a 1:10 dilution in distilled H2O (0.05 M Tris buffer, pH 7.7)
2. Tris buffer containing physiological ions
xe2x80x83b. Dilute 1:10 in distilled H2O. This yields 0.05 M Tris hydrochloride pH 7.7; containing sodium chloride (120 mM), potassium chloride (5 mM), calcium chloride (2 mM) and magnesium chloride (1 mM).
3. [4-3H]-Clonidine Hydrochloride (20-30 Ci/mmol) is obtained from New England Nuclear. For IC50 determinations: 3H-Clonidine is made up to a concentration of 120 nM and 50 xcexcl added to each tube (yields a final concentration of 3 nM in the 2 ml volume assay).
4. Clonidine hydrochloride is obtained from Boehringer Ingelheim. A stock solution of 0.1 mM clonidine is made up to determine nonspecific binding. This yields a final concentration of 1 xcexcM in the assay (20 xcexcl to 2 ml).
5. Test compounds. For most assays, a 1 mM stock solution is made up in a suitable solvent and serially diluted, such that the final concentration in the assay ranges from 10xe2x88x925 to 10xe2x88x928M. Seven concentrations are used for each assay and higher or lower concentrations may be used, depending on the potency of the drug.
B. Tissue Preparation
Male Wistar rats are sacrificed by decapitation and the cortica tissue rapidly dissected. The tissue is homogenized in 50 volumes of 0.05 M Tris buffer, pH 7.7 (buffer 1b) with the Brinkman Polytron, then centrifuged at 40,000 g for 1 minutes. The supernatant is discarded and the pellet rehomogenized in the original volume of 0.05 M Tris buffer, pH 7.7 and recentrifuged as before. The supemate is discarded and the final pellet rehomogenized in 50 volumes of Buffer 2b. This tissue suspension is then stored on ice. The final tissue concentration is 10 mg/ml. Specific binding is 1% of the total added ligand and 80% of total bound ligand.
C. Assay 100 xcexcl 0.5 M Tris-physiological salts, pH 7.7 (buffer 2a)
Tissue homogenates are incubated for 20 minutes at 25xc2x0 C. with 3 nM 3H-clonidine and varying drug concentrations, then immediately filtered under reduced pressure on Whatman GF/B filters. The filters are washed with three five ml volumes of ice-cold 0.05 M Tris buffer, pH 7.7, then transferred to scintillation vials. Ten ml of liquescent counting solution is added to each sample which is then counted by liquid scintillation spectroscopy. Specific clonidine is defined as the difference between total bound and that performed using log-probit analysis. The percent inhibition at each drug concentration is the mean of triplicate determinations.
Depression treatment is achieved when the present aminoalkylphenol derivatives and related compounds are administered to a subject requiring such treatment as an effective oral, parenteral or intravenous dose of from 0.10 to 50 mg/kg of body weight per day. A particularly effective amount is about 10 mg/kg of body weight per day. It is to be understood, however, that for any particular subject, specific dosage regimens should be adjusted according to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compound. It is to be further understood that the dosages set forth herein are exemplary only and that they do not, to any extent, limit the scope or practice of the invention.
Acetylcholinesterase inhibitors and clonidine binding inhibitors are known in the art as being useful as relievers of memory dysfunction and as antidepressants, respectively. (See V. Kumar in Alzheimer""s Disease: Therapeutic Strategies, E. Giacobini and R. Becker Eds.; Birkhauser, Boston 1994 for memory dysfunction utility and K. F. Tipton in Biochemical and Pharmacological Aspects of Depression, K. F. Tipton and U. B. H. Youdin, Eds., Taylor and Francis, London 1989, for antidepressant utility.
Depression frequently attends memory dysfunction associated with Alzheimer""s disease and responds to antidepressant intervention. Thus, the antidepressant component of the pharmacological properties of the compounds of the present invention provide both desirable effects in one chemical entity, providing both therapies in one administration, where indicated. See, for example, W. W. Pendlebury and P. R. Solomon, Neurobiology of Aging, 15, 287 (1994) at page 287, among others.
Effective amounts of the compounds of the invention may be administered to a subject by any one of various methods, for example, orally as in capsules or tablets, parenterally in the form of sterile solutions or suspensions, and in some cases intravenously in the form of sterile solutions. The free base final products, while effective themselves, may be formulated and administered in the form of their pharmaceutically acceptable addition salts for purposes of stability, convenience of crystallization, increased solubility and the like.
Preferred pharmaceutically acceptable addition salts include salts of mineral acids, for example, hydrochloric acid, sulfuric acid, nitric acid and the like, salts of monobasic carboxylic acids such as, for example, acetic acid, propionic acid and the like, salts of dibasic carboxylic acids such as, for example, maleic acid, fimaric acid, oxalic acid and the like, and salts of tribasic carboxylic acids such as, for example, carboxysuccinic acid, citric acid and the like.
The active compounds of the present invention may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the aforesaid compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 0.5% of active compounds, but may be varied depending upon the particular form and may conveniently be between 4% to about 75% of the weight of the unit. The amount of present compound in such composition is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are prepared so that an oral dosage unit form contains between 1.0-300 mgs of active compound.
The tablets, pills, capsules, troches and the like may, also contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin or a flavoring agent such as peppermint, methyl, salicylate, or orange flavoring may be added. When the dosage unit is a capsule it may contain, in addition to materials of the above type, a liquid carrier such as fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
For the purposes of parenteral therapeutic administration, the active compounds of the invention may be incorporated into a solution or suspension. These preparations should contain at least 0.1% of the aforesaid compound, but may be varied between 0.5 and about 50% of the weight thereof. The amount of active compound in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.5 to 100 mgs of the active compound.
The solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agepts such as benzyl alcohol or thyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparations can be enclosed in ampules, disposable syringes or multiple vials made of glass or plastic.